Electroporation device and injection apparatus

ABSTRACT

An apparatus is provided for injecting a fluid into body tissue, the apparatus comprising: a hollow needle; and fluid delivery means, wherein the apparatus is adapted to actuate the fluid delivery means in use so as to automatically inject fluid into body tissue during insertion of the needle into the said body tissue.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/048,314, filed Feb. 19, 2016, which is a continuation of U.S.application Ser. No. 11/985,825, filed Nov. 16, 2007, which is acontinuation of U.S. application Ser. No. 10/612,304, filed Jul. 3, 2003(now issued as U.S. Pat. No. 7,328,064), which claims the benefitprovided in 35 USC Section 119, of U.K. Patent Application No.GB021552919, filed on Jul. 4, 2002, and to U.K. Patent Application No.GB 0215523.2, filed on Jul. 4, 2002, the entire contents of each ofwhich are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to the injection of substances into tissueand, in one preferred application, to delivery by electroporation, i.e.the process of introducing substances into cells during or after theapplication of an electric field. More particularly, the presentinvention relates to a device which may be used in delivery byelectroporation.

BACKGROUND

Electroporation is used for example in the treatment of cancer or ingene therapy. Electroporation provides a method of deliveringpharmaceuticals or nucleic acids (e.g. DNA) into cells, e.g. skeletalmuscle cells. Thus for example the muscle may be electrically stimulatedat the same time or shortly after the pharmaceutical or DNA is injected.This method works on the principle that cells act as an electricalcapacitor generally unable to pass current. Subjecting the cells to anelectric field creates transient permeable structures or micropores inthe cell membrane. The permeability or the pores are large enough toallow the pharmaceuticals and/or DNA to gain access to the cells. Withtime, the pores in the cell membrane close and the cell once againbecomes impermeable.

Various devices for effecting electroporation have been suggested. U.S.Pat. No. 6,208,893 discloses an electrode template apparatus having aplurality of bores through which a plurality of needle electrodesextend, each bore being separately connected to a conductor so that eachof the electrodes can be connected to a power supply in use. Aninsulating portion can be provided along the midportion of eachelectrode so as to isolate the body tissue adjacent the insulated partof the needle from the electric field produced by the electrode in use.Further, one or more of the needle electrodes may be hollow and caninclude openings through which medicinal substances can be injected intothe body tissue.

EP0693951B discloses a device for the implementation ofelectrochemotherapy. The device comprises electrode needles throughwhich electric pulses are applied. The electrode needles are hollow soas to allow active substances to be injected locally into the bodytissue to be treated. Holes can be provided along the length of theneedles as well as at the ends thereof to improve the distribution ofinjected substances. An insulating sheath can also be provided over apart of the needle lengths as a means of preventing the application ofelectrical pulses to certain zones.

The present invention at least in its preferred embodiments seeks toprovide a device which can be used in electroporation in vivo, inparticular in gene therapy.

One problem in electroporation is that DNA is injected intra-muscularlyand may become trapped between muscle bundles or in adipose tissuebetween muscle cells. Further, the DNA can be stopped by tendons orother connective tissue barriers. This will make it difficult to obtainan even distribution of DNA over the entire area of tissue to which anelectric field is to be applied. It is important to match the volumecovered by the electric field applied during electroporation to the siteof DNA injection to limit the distribution of the electrical field orvolume of DNA. An additional problem is that when carried out on humanbeings, injections of large volumes of fluid at one site may causeconsiderable pain to the patient.

SUMMARY

From a first aspect, the present invention provides an apparatus forinjecting a fluid into body tissue, the apparatus comprising: a hollowneedle; and fluid delivery means, wherein the apparatus is adapted toactuate the fluid delivery means in use so as to concurrently(preferably automatically) inject fluid into body tissue duringinsertion of the needle into the said body tissue. This has theadvantage that the ability to inject the fluid gradually while theneedle is being inserted leads to a more even distribution of the fluidthrough the body tissue. It is also believed that the pain experiencedduring injection is reduced due to the distribution of the volume offluid being injected over a larger area.

In addition, the automatic injection of fluid facilitates automaticmonitoring and registration of an actual dose of fluid injected. Thisdata can be stored by a control unit for documentation purposes ifdesired.

It will be appreciated that the rate of injection could be either linearor non-linear and that the injection is preferably carried out after theneedles have been inserted through the skin of the subject to be treatedand while they are inserted further into the body tissue.

Suitable tissues into which fluid may be injected by the apparatus ofthe present invention include tumour tissue, skin or liver tissue butwill preferably be muscle tissue.

Preferably the apparatus further comprises needle insertion means forguiding insertion of the needle into the body tissue. Still morepreferably, the rate of fluid injection is controlled by the rate ofneedle insertion. This has the advantage that both the needle insertionand injection of fluid can be controlled such that the rate of insertioncan be matched to the rate of injection as desired. It also makes theapparatus easier for a user to operate.

If desired means for automatically inserting the needle into body tissuecould be provided.

A user could choose when to commence injection of fluid. Ideallyhowever, injection is commenced when the tip of the needle has reachedmuscle tissue and the apparatus preferably includes means for sensingwhen the needle has been inserted to a sufficient depth for injection ofthe fluid to commence. This means that injection of fluid can beprompted to commence automatically when the needle has reached a desireddepth (which will normally be the depth at which muscle tissue begins).The depth at which muscle tissue begins could for example be taken to bea preset needle insertion depth such as a value of 4 mm which would bedeemed sufficient for the needle to get through the skin layer.

In one preferred embodiment the sensing means comprises an ultrasoundprobe.

In an alternative preferred embodiment the sensing means comprises meansfor sensing a change in impedance or resistance. In this case, the meansmay not as such record the depth of the needle in the body tissue butwill rather be adapted to sense a change in impedance or resistance asthe needle moves from a different type of body tissue into muscle.Either of these alternatives provide a relatively accurate and simple tooperate means of sensing that injection may commence. The depth ofinsertion of the needle can further be recorded if desired and could beused to control injection of fluid such that the volume of fluid to beinjected is determined as the depth of needle insertion is beingrecorded.

The apparatus preferably further comprises: a base for supporting theneedle; and a housing for receiving the base therein, wherein the baseis moveable relative to the housing such that the needle is retractedwithin the housing when the base is in a first rearward positionrelative to the housing and the needle extends out of the housing whenthe base is in a second forward position within the housing. This isadvantageous for a user as the housing can be lined up on the skin of apatient, and the needles can then be inserted into the patient's skin bymoving the housing relative to the base.

As stated above, it is desirable to achieve a controlled rate of fluidinjection such that the fluid is evenly distributed over the length ofthe needle as it is inserted into the skin. Preferably therefore, thefluid delivery means comprise piston driving means adapted to injectfluid at a controlled rate.

The piston driving means could for example be activated by a servomotor. Preferably however, the piston driving means are actuated by thebase being moved in the axial direction relative to the housing.

It will be appreciated that alternative means for fluid delivery couldbe provided. Thus, for example, a closed container which can be squeezedfor fluid delivery at a controlled or non-controlled rate could beprovided in the place of a syringe and piston system.

The apparatus described above could be used for any type of injection.It is however envisaged to be particularly useful in the field ofelectroporation and so it preferably further comprises means forapplying a voltage to the needle. This allows the needle to be used notonly for injection but also as an electrode during, electroporation.This is particularly advantageous as it means that the electric field isapplied to the same area as the injected fluid. There has traditionallybeen a problem with electroporation in that it is very difficult toaccurately align an electrode with previously injected fluid and souser's have tended to inject a larger volume of fluid than is requiredover a larger area and to apply an electric field over a higher area toattempt to guarantee an overlap between the injected substance and theelectric field. Using the present invention, both the volume of fluidinjected and the size of electric field applied may be reduced whileachieving a good fit between the electric field and the fluid.

As an aid to medical staff who may treat a large number of patients in aday, the apparatus may further comprise means for recording the identityof a subject to be treated and data from a treatment process.

Further, a fluid dispense vessel may be provided for use in theapparatus of the invention, in which a bar-code is provided on thevessel to identify the contents thereof. This barcode could berecognised by a pulse generator used in electroporation which would beprogrammed to automatically set up the required injection speed andelectroporation conditions for the bar code.

From a further aspect, the present invention provides a method ofinjecting a fluid into body tissue, the method comprising: injecting thefluid into the body tissue through a hollow needle while the said needleis being inserted into the said body tissue. The injection of fluidgradually while the needle is being inserted leads to a more evendistribution of the fluid through the body tissue. It is also believedthat the pain experienced during injection is reduced due to thedistribution of the volume of fluid being injected over a larger area.

Preferably, the needle tip is first inserted into the skin and injectionis then carried out while the needle is inserted further into the bodytissue.

Still more preferably, the injection is commenced when the needlereaches a first desired depth in the body tissue and is stopped when theneedle reaches a second desired depth in the body tissue.

The method of injection described above may advantageously be used inconjunction with a method of electroporation wherein fluid is injectedinto body tissue by the method of injection of the invention and avoltage is then applied to the needle.

The method of injection described above may advantageously be used inconjunction with an alternative method of electroporation wherein fluidis injected into body tissue by the method of injection of theinvention, the needle is withdrawn from the body tissue, an electrode isinserted in the place of the needle, and a voltage is applied to theelectrode.

Gene therapy by electroporation involves administering a dose of betweenabout 10 .mu.L and 10 ml (e.g. between 10 .mu.L and 1 ml, preferablybetween 100 .mu.L and 1 ml) of DNA solution. DNA is toxic if too much isincorporated into cells and so the quantity of DNA in solution must notbe too high. Thus, the quantities of solution are relatively small and,especially in larger animals such as human beings, it is difficult toadminister both DNA and electric field to the right place in the muscle.Further, as the cells being treated should not be damaged, theelectroporation device should be much gentler than the prior art deviceswhose primary use is in the treatment of cancer where the treated cellsare killed. Ideally therefore, the electroporation device should notproduce undue fields and should also not include any relatively blunt orbulky tissue piercers.

From a first aspect, the present invention provides an electroporationdevice comprising: a needle for injecting a substance into body tissue;and an insulating sheath adapted to surround the needle and having oneor more apertures formed along the length thereof through which theelectric field may propagate in use, wherein the needle is axiallymoveable relative to the sheath.

The device of the invention has the advantage that if the needle is alsoused as an electrode, as the needle is axially moveable relative to thesheath, the needle can be withdrawn so that the insulating sheathcompletely surrounds the needle after the device has been inserted intothe body tissue and before the electric field generating means areactivated. Thus in use, the electric field propagates through theapertures in the sheath, and so the formation of uneven electric fieldstrengths in the body tissue to be treated is avoided as no edge effectsare created.

Preferably, the needle for injecting a substance into body tissue alsoconstitutes an electrode via which an electric field is propagated inuse. Thus, in this preferred embodiment, the needle is connectable to avoltage source. It will of course be appreciated that, in oneembodiment, the needle could remain connected to the voltage source atall times.

However if necessary, the device may be adapted to allow the needle tobe removed from the insulating sheath after injection of the substanceinto the body tissue so that the needle can be replaced by an electroderod prior to activation of the electric field. This would beadvantageous for example to avoid the release of unwanted metal ions bythe needle which could be caused by the provision of an electric chargeon the needle. In this embodiment, the electrode rod would be arrangedso as to be completely surrounded by the sheath in use so that again, noedge effects would be produced by the electric field in use

The sheath could be formed of any electrically insulating andbiologically compatible material. Preferably however, the sheath isformed from polytetrafluoroethylene (TEFLON®).

Any number of apertures could be provided in the insulating sheath. Inone preferred embodiment, the apertures are provided along one axiallyextending line on the sheath only. In an alternative preferredembodiment, the apertures are provided so as to be spaced around thecircumference of the sheath. The actual number and arrangement ofapertures provided in the sheath will depend on the electric fieldpatterns required in the tissue to be treated.

The apertures in the insulating sheath could be formed in a number ofways such as but not limited to: cutting through the sheath, pushing theapertures out or laser ablation. Where apertures are required on oneside only of the sheath, during aperture formation a rod can be providedwithin the sheath to prevent holes forming on both sides.

The electroporation device of the invention could be used alone.Preferably however, two or more electroporation devices are usedtogether and if required, any number of the devices could be used Thusfor example, a group of four, six or eight devices could be used. Whereone or more devices are used, the needles and sheaths can be mounted toextend downwardly through a block in which they are arranged adjacent toone another. Consequently, it will be appreciated that any number ofneedles (i.e 1 or more could be used).

Preferably, means are provided such that in use the depth of insertionof a needle is determined and injection of a substance into the bodytissue to be treated is commenced when the needle has reached a desireddepth.

This is believed to be novel and inventive in its own right and so froma further aspect the present invention provides a device comprising aneedle for injection of a substance into body tissue, and means forsensing the depth of insertion of the needle and commencing injection ofa substance via the needle when a desired depth has been reached.

Various means could be provided to determine that the needle has reacheda desired depth for injection to commence. For example, means fordetermining the electrical resistance of the tissue which will varydepending on tissue type (dermis, fat or tissue) could be provided.Preferably however, a moveable contact can be provided on the devicesuch that in use, the contact determines when the needle has beeninserted to a sufficient depth into the body tissue to be treated andthen causes injection of a substance to commence. This allows automaticinjection of a substance to commence when the needle reaches the correctdepth in the body tissue to be treated. The injection can be carried outeither while the needle is stationary or while it is continuing to beinserted.

Still more preferably, the moveable contact further determines when theneedle has been inserted to the maximum depth at which injection shouldbe carried out and then causes injection of the substance to stop. Inthis way it is possible for the substance to be automatically injectedover the height of tissue over which an electric field will be producedin use.

Viewed from a further aspect the invention provides a method ofelectroporetic treatment of a human or nonhuman animal (e.g. a mammal,bird or reptile), said method comprising inserting the needle of adevice according to the invention into tissue (e.g. muscle tissue) insaid animal, injecting an active agent (e.g. a pharmaceutical or nucleicacid) through the needle into the tissue, withdrawing the needle suchthat the tip thereof is within the sheath, and applying an electricfield between the needle and an electrode.

It will be appreciated that the electrode could be provided by theneedle of a second device according to the invention disposed inside afurther sheath. Alternatively, the electrode could be a different typeof electrode which had been inserted into the body tissue or anelectrode which had been applied to the skin surface.

Viewed from a still further aspect the invention provides a method ofelectroporetic treatment of a human or non-human animal (e.g. a mammal,bird or reptile), said method comprising inserting the needle of adevice according to the invention into tissue (e.g. muscle tissue) inthe animal, injecting an active agent (e.g. a pharmaceutical or nucleicacid) through the needle into the tissue, withdrawing the needle fromthe sheath, inserting a first electrode into the sheath such that thetip of the first electrode does not extend out of the sheath into thetissue, and applying an electric field between the first electrode and asecond electrode.

It will be appreciated that the second electrode could be provided bythe needle of a second device according to the invention disposed insidea sheath. Alternatively, the electrode could be a different type ofelectrode which had been inserted into the body tissue or an electrodewhich had been applied to the skin surface.

The device according to the invention could for example be used in themethod of WO98/43702, the contents of which are herein incorporated byreference. Preferably, the device would be used with a square bipolarelectric pulse.

In the device of U.S. Pat. No. 6,208,893 as discussed above, the needleelectrodes are inserted axially from above into the respective bores inuse and are removed by being drawn axially outward after use. Thepresent inventors have identified a problem with the use of such adevice in which the bores become contaminated with the blood of ananimal or person when the needles are withdrawn after use as the tips ofthe needles pass through the bores. Thus, the apparatus can only bereused after very thorough disinfection which is time consuming andexpensive.

From a further aspect, the present invention seeks to provide a devicewhich overcomes this problem. In a first aspect, the present inventionprovides a device for use in electroporation comprising a housing formedin two or more parts, wherein the parts are moveable relative to oneanother to open and close the housing, and a groove is formed in asurface of at least one of said parts in such a way as to form a boreextending through the housing when the housing is closed. Preferably thebore is adapted to receive a needle in use and the needle can beinserted and removed from the bore by opening the housing.

Thus, as the needle can be removed from the bore by opening the housingand so lifting it out of an open groove, there is no need to remove theneedle from the bore by pulling it out in the axial directionConsequently blood and any other bodily fluids left on the tip of theneedle after use need not be brought through the bore and so the housingwill not be contaminated as in the prior art devices.

The parts of the housing could for example be held together in theclosed position by a removable belt extending around the outside of thehousing. Preferably however, the parts are hingedly attached to oneanother. This has the advantage of making the housing particularly easyto open and close.

The housing could for example be formed in four parts which make up thequarters of a cuboid, each part having a groove with the cross sectionof a quadrant formed at the inner comer thereof. Alternatively, thehousing could be formed in two parts, with a groove having for example asemi-circular or square cross section formed on the inner surface of onepart while the surface of the other part is flat. Preferably however thehousing is formed of two parts, a groove of semicircular cross sectionbeing provided on the inner surface of each part and being positioned toform a bore of circular section from 10 the two grooves when the housingis closed. It will be appreciated that in this arrangement, the parts ofthe housing can be hingedly attached together at one end thereof in amanner allowing simple manufacture and use of the device. Further, thecircular cross section of the bore is particularly advantageous as theneedles to be held therein are normally circular in cross section.

Still more preferably, the housing is formed to receive two needles intwo respective bores. Although the device could be used with any numberof needles, two needles are often required to carry out electroporationand so this is a particularly preferred arrangement.

The needles could be connected to an electric power supply by standardmeans such as cables attached to an end of the needle extending out ofthe housing. Preferably however an electrical contact is provided for orwithin the or each bore so that a needle within the bore is brought intocontact with an electrical power supply when the housing is closed. Thishas the advantage that a user need not spend time connecting a needle toa power supply by attaching cables etc. and so is much quicker andsimpler to use.

Still more preferably, the device is configured so as to lock the needlein position within the bore when the housing is closed in use. Thus, noadditional means need be provided to stop the needle from movingrelative to the housing during insertion of the needle into the bodytissue to be treated and the subsequent electroporation process.

In one preferred embodiment, a foot pedal could be provided to activatethe power supply when required for electroporation. This has theadvantage that a user would have their hands free at all times to holdthe device and the needle(s) in place in an animal or person beingtreated. It will be appreciated however that alternative means such as aswitch provided on the needle holder could be provided for activatingand deactivating the power supply.

The device of the invention could be used with any standard known,approved needles and injection assemblies or syringes.

In one preferred embodiment, the device could be used with one or moreneedles, wherein each said needle is surrounded by an insulating sheath,the sheath having one or more apertures formed along the length thereof.The use of such insulated needles has the advantage of reducing theproduction of edge effects when the needle is used as an electrode.

Preferably, the same needle is used for injecting a substance into thebody tissue to be treated and applying an electric field. Wherenecessary however, the needle could be withdrawn front the sheatharranged within a bore of the housing after injection of a substanceinto the body tissue to be treated and substituted by an electrode rodfor carrying out the electroporation. This would be advantageous forexample to avoid the release of unwanted metal ions by the needle whichcould be caused by the provision of an electric charge on the needle. Inthis embodiment, the electrode rod could be arranged to be completelysurrounded by an insulating sheath to avoid the production of edgeeffects by the electric field in use. Further, the insulating sheatharranged within the bore would protect the bore from contamination byblood and/or other bodily fluids as the needle was withdrawn axiallyfrom within the bore and sheath.

Preferably, even if the needle is not completely withdrawn from thesheath after injection of a substance into the body tissue, the needleis still axially moveable relative to the sheath. This allows the needleto be withdrawn inside the sheath after injection so that it is fullysurrounded by the sheath before the application of an electric field.This has the advantage of further reducing the production of edgeeffects by the electric field in use.

The sheath could be formed of any electrically insulating andbiologically compatible material. Preferably however, the sheath isformed from polytetrafluoroethylene (TEFLON®).

Preferably, the needles used for injection of a substance into the bodytissue to be treated are attached to syringe devices via which injectionis carried out. It would also be possible however for the needles to beprovided separately for attachment to injection means at an appropriatetime.

Preferably, the device is provided with means for determining the depthof insertion of a needle into the body tissue to be treated in use andfor automatically commencing injection of a substance into the bodytissue to be treated when a desired depth of the needle has beenreached.

Preferably a moveable contact can be provided on the device such that inuse, the contact determines when the needle has been inserted to asufficient depth into the body tissue to be treated and then causesinjection of a substance to commence. This allows automatic injection ofa substance to commence when the needle reaches the correct depth in thebody tissue to be treated. The injection can be carried out either whilethe needle is stationary or while it is continuing to be inserted.

Still more preferably, the moveable contact further determines when theneedle has been inserted to the maximum depth at which injection shouldbe carried out and then causes injection of the substance to stop. Inthis way it is possible for the substance to be automatically andaccurately injected over the height of tissue over which an electricfield will be produced in use.

Viewed from a further aspect, the present invention provides a method ofelectroporation treatment of a human or non-human animal (e.g. a mammal,bird or reptile), said method comprising inserting a needle held in adevice according to the invention into tissue (e.g. muscle tissue) insaid animal, injecting an active agent (e.g. a pharmaceutical or nucleicacid) through the needle into the tissue, applying an electric fieldbetween the needle and an electrode, removing the needle from the tissueand opening the housing of the device to remove the needle therefrom.

Preferably, the needle could be pushed further into the tissue afterinjection and before the application of an electric field to enable theelectric field to be applied over the full height of injected fluid.

It will be appreciated that the electrode could be provided by a secondneedle held in a or the device according to the invention.Alternatively, the electrode could be a different type of electrodewhich had been inserted into the body tissue or an electrode which hadbeen applied to the skin surface.

It will further be appreciated that the needle could be any knownapproved form of needle or any other type of needle described herein.

In an alternative preferred method of treatment, the needle is removedfrom the device according to the invention after injection and replacedby an electrode, an electric field being applied between the twoelectrodes.

The device according to the invention could for example be used in themethod of WO 98/43702, the contents of which are herein incorporated byreference. Preferably, the device would be used in an electroporationmethod in which a square uni or bipolar electric pulse is applied to theelectrode.

From a further aspect, the present invention provides a method ofdetermining when a needle has been inserted to a desired depth in bodytissue comprising measuring a change in impedance as the needle isinserted into the body tissue.

Although this could be achieved in various ways, two needles arepreferably inserted into the body tissue adjacent one another and theimpedance between the needles is measured.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention will now be described, by way ofexample only, and with reference to the accompanying drawings in which:

FIG. 1 is a schematic side elevation view of an electroporation deviceaccording to a first embodiment of the invention;

FIGS. 2a to 2c are schematic side elevation views showing three stagesin the operation of an electroporation device according to the firstembodiment of the invention including a skin contact device;

FIG. 3 is a perspective view of an electroporation device according to asecond embodiment of the invention in an open position;

FIG. 4 is a perspective view of the device of FIG. 3 in the closedposition;

FIG. 5 is a schematic plan view of a part of the device of FIG. 3holding a needle and injection device;

FIG. 6 is a schematic elevational view of an alternative needle andinjection device for use with the device of FIG. 3;

FIG. 7 is a side perspective view of an electroporation device accordingto a third embodiment of the invention;

FIG. 8 is an underneath perspective view of the device of FIG. 7;

FIG. 9 is a side perspective view of the base of the device of FIG. 7;

FIG. 10 is a side elevational view of the base of the device of FIG. 7;

FIG. 11 is a top plan view of the base of the device of FIG. 7;

FIG. 12 is a side elevational view of the base of the device of FIG. 7from the opposite side to that shown in FIG. 10;

FIG. 13 is a cross sectional side view of the cover of the device ofFIG. 7;

FIG. 14 is a side view of the device of FIG. 7 when fully assembledready to start the process;

FIG. 15 is a side view of the device of FIG. 7 at the point at which theneedles have penetrated the skin, ready to start the injection andneedle insertion process;

FIG. 16 is a side view of the device of FIG. 7 halfway during needleinsertion;

FIG. 17 is a side view of the device of FIG. 7 when needle insertion andinjection have been completed (i.e. when the device is ready forelectroporation to be carried out, before the needles are withdrawn);

FIG. 18a is an exploded view of the gear mechanism of the device of FIG.7 for driving the needle insertion and injection process;

FIG. 18b is a view of the gear mechanism of FIG. 18a mounted on the baseunit;

FIG. 18c is a view of the base unit showing the gear mechanism of FIG.18a and the rack member in place.

FIG. 19 shows the amount of SEAP used in serum in a test using a deviceaccording to the invention; and

FIGS. 20a and 20b shows the results of the test using abeta-galactosidase expressing vector introduced using a device accordingto the invention.

DETAILED DESCRIPTION OF ILLUSTRATED EMBODIMENTS

As shown in FIG. 1, an electroporation device according to a firstembodiment of the invention comprises two separate needle assemblies 2mounted adjacent to one another in a support block 4. Each needleassembly 2 comprises a hollow needle 6 having a sharp end 8 which isopen to allow the injection of fluids via the opening. The other end ofeach of the needles 6 is connected to a fluid holding chamber 10 havinga piston 12 arranged therein so as to form a syringe arrangement forinjecting fluid via the needles in use. These syringes may be standardsingle-use syringes.

First and second electrically insulating sheaths 14 made of TEFLON® andhaving a greater cross sectional diameter than that of the needles 6 arearranged to extend around the needles 6. Three apertures 16 spaced apartin the axial direction are provided along the length of each sheath 14.The device is configured so as to allow axial movement of the needles 6relative to the sheaths 14.

A voltage supply 18 is provided on the support block 4 which can beconnected and disconnected from the needles 6 of the electroporationdevice.

In use, a required dose of DNA (which could for example be 100 .mu.L) isprovided in each of the fluid holding chambers 10 and the needles 6 areinserted into the skin of an animal or person to be treated. It isadvantageous that the volume of fluid for injection should be small asthis will insure that the injected fluid is kept close to the shaft ofthe needle (i.e. will be kept within a high electric field strength zoneduring electroporation). At this stage, the sharp ends 8 of the needles6 extend beyond the TEFLON® sheaths 14 and so provide a sharp point forpiercing the skin and penetrating into the muscle or body tissue to betreated. During insertion, the relative position of the needles 6,sheaths 14 and support block 4 does not vary as the elements are lockedinto place relative to one another. The needles are then insertedfurther until they reach the correct depth in the muscle or other bodytissue to be treated. Once they have reached this depth and while stillbeing inserted, the DNA is injected into the muscle by pushingdownwardly on the pistons 12 to empty the fluid holding chambers 10. Ifnecessary, the needles can then be pushed further down into the muscleafter injection. This ensures that the needles acting as electrodescover the area into which the fluid has been injected.

After insertion of the needles and once the DNA has been injected, theneedles 6 are withdrawn slightly (i.e. moved axially towards the supportblock 4) relative to the TEFLON® sheaths 14 which remain in theiroriginal inserted position. Thus, the sharp ends 8 of the needles 6 areretracted to locate within the TEFLON® sheaths 14. Once the needles 6have been retracted as described, the voltage source 18 is activated andelectroporation proceeds with each of the needles 6 acting as anelectrode. The electric field produced by the needles 6 acting aselectrodes propagates into the muscle or body tissue to be treated viathe apertures 16 formed along the length of the TEFLON® shields 14. Thishas the advantage that no unwanted edge effects are created in themuscle or body tissue to be treated.

In a further improvement to the device of FIG. 1 (as shown in FIGS. 2ato 2c ), means are provided to sense when during insertion the needles 6are at the correct depth in the muscle or body tissue for injection ofthe DNA to begin and to automatically move the pistons 12 to effect theinjection. These means comprise a moveable skin contact 20 whichcontacts the skin S as shown in FIGS. 2a to c . As the needles 6 areinserted into the muscle or body tissue to be treated, the contact 20 ispushed upwardly towards the support member 4. The contact member 20 isattached to a lever mechanism consisting of a substantially verticallink 22 extending upwardly from the contact member 20 and a lever 24which is attached at a first end to the vertical link 22. The lever 24is attached at its other end to means 26 for causing the pistons 12 tomove downwardly. The lever is adapted to pivot about a point 28 on thesupport member 4 located between the two ends of the lever 24. Thus, asthe contact 20 moves upwardly relative to the support member 4 in use,the lever 24 pivots causing the piston moving means 26 to push thepistons down gradually so as to effect injection of the fluids over theheight of the needles being inserted. As shown, the piston moving meanscomprise a vertical member 27 attached to the lever 24 so as to movedownwardly as the lever pivots and a cross piece 30 attached to theother end of vertical member 27 which acts to push the pistons down asit moves downwardly with the vertical member.

The relative location of the skin contact 20 and lever mechanism can beadjusted to ensure injection of the fluids once the needles have reachedthe muscle tissue and while they are being inserted further into thetissue to ensure a uniform distribution of sample in the area around theelectrodes in the muscle.

FIG. 2a shows the device before the pistons have been pushed down withthe tips of the needles just inserted into the skin. FIG. 2b shows thedevice when the needles are fully inserted to the required depth in themuscle tissue and the pistons 12 have been fully depressed by the actionof the lever mechanism. FIG. 2c shows the device once the needles havebeen attached to a power supply 18 after injection of the fluids. Asshown, the syringes have been removed although this is not essential.

In alternative embodiments, lasers or sensors could be used to detectthe depth of insertion of the needles and automatically initiateinjection of the fluids at a desired depth instead of the mechanicalskin contact arrangement described above.

The contact or sensors can be further adapted to sense when the needles6 have reached a depth in the body tissue at which injection of thefluids should stop so as to ensure that fluid is only injected into theheight of body tissue to which an electric field will be applied in use.

It will be appreciated that one advantage of the embodiment of theinvention described above is that known cannula devices which arealready on the market and so have marketing approval can be used toprovide the needle and sheath assemblies of the device, the onlymodification which is required being the formation of the apertures 16in the sheaths. Thus, the use of such commercially available cannulascan ensure rapid and inexpensive regulatory clearance. One example of aknown cannula device which could be used is the 0.8/25 mm diameterVENFLON® sold by BOC Ohmeda AS of Helsingborg, Sweden.

In an alternative embodiment of the invention (not shown) the needles 6can be withdrawn from the muscle or body tissue to be treated after theDNA has been injected into it and electrodes having a similar shape butmade of an alternative metal such as stainless steel can be insertedbefore electroporation is carried out. This could be useful for examplein a situation where biologically incompatible metal ions would beemitted if the needles 6 were also used as the electrodes.

As shown in FIG. 3, a device according to a second embodiment of theinvention comprises a housing 41 made up of two halves 42, 44 which arejoined together by a hinge 46 Each half 42, 44 of the housing is arectangular solid and the hinge 46 is provided between adjacent endfaces thereof so that the upper plane rectangular surfaces of each halfof the housing can be pivoted towards each other until the upper surface48 of the first half 42 lies directly above the upper surface 50 of thesecond half 44. In this position, the housing is said to be closed andthis is shown in FIG. 4.

From FIG. 3, it can be seen that recesses or grooves are formed in theupper surfaces 48, 50 of each of the two halves 42, 44. Each groove issemi-circular in cross section and has a wider portion 52 extending froma first side 54 of the housing half which leads into a narrower portion56 which extends to the other side 58 of the housing half. Thus, in usethe needle 60 of a syringe device fits into the narrower portion 56while the syringe or injection part 62 adjacent the needle fits into thewider portion 52 as shown in FIG. 5.

The upper surface 48 of the first half 42 of the housing 41 has tworecesses of the type described above formed therein which are laterallyspaced from one another. Two recesses are also formed in the uppersurface 50 of the second half 44 at corresponding locations such that,when the housing is closed so that the first 48 and second 50 surfacesare arranged one above the other, the recesses in the first and secondsurfaces join to form two bores 63 within which respective needles andsyringe or injection devices may be held.

Also as shown in FIG. 3, an electrical contact element 64 is provided inthe narrower part 56 of each recess in the first half 42 of the housing.The electrical contact elements 64 are connected to an electrical powersource V and arranged so that a needle placed within the recess willautomatically be brought into contact with the electrical contactelement when the housing is closed.

The device shown and described with reference to FIGS. 3 and 4 can beused with any standard approved needle and syringe device such as forexample the Sterile EO CE0123, STERICAN® 0.40.times.40 rom BL/LB,27G.times.11/2.

In an alternative embodiment, the device can be used with syringedevices including needles 6 which are surrounded by insulating sheaths14 such as those shown in FIG. 1 for use with the device of the firstembodiment of the invention. A syringe device of this type for use inthe second embodiment of the invention is shown in FIG. 6. As can beseen, the device includes a needle 6 and a TEFLON® sheath 14. As shownin FIG. 6, the insulating sheath 14 which surrounds the needle has threeapertures 16 spaced apart from one another in the axial direction andprovided along the length of the sheath. A fluid container 10 includinga piston 12 is provided at one end of the needle for injecting fluidtherethrough. In one embodiment, the needle is axially moveable relativeto the sheath so that after it has been inserted into the body tissue tobe treated, the needle is withdrawn into the sheath. This avoids theformation of harmful edge effects when an electric field is applied tothe needle. Known cannula devices which are already on the market and sohave marketing approval can be used to provide the needle and sheathassemblies of the device, the only modification which is required beingthe formation of the apertures 16 in the sheaths. Thus, the use of suchcommercially available cannulas can ensure rapid and inexpensiveregulatory clearance. One example of a known cannula device which couldbe used is the 0.8/25 mm diameter VENFLON® sold by BOC Ohmeda AB ofHelsingborg, Sweden.

If desired, means may be provided with the device of the secondembodiment of the invention to sense when the needles 6, 60 are at thecorrect depth in the muscle or body tissue for injection of the DNA tobegin and to automatically move the pistons 12 to effect the injectionin the same way as for the first embodiment of the invention as shown inFIGS. 2a to 2c . When used with the device of the second embodimenthowever, the lever 24 pivots about point 28 on the housing 41 ratherthan support block 4.

A method of electroporation treatment using the device of FIGS. 3 and 4will now be described. This method could be carried out on any human ornon-human animal. A required dose of DNA (which could for example be 100.mu.!) is provided in each fluid container 12,62. Then the syringedevices are inserted into respective recesses 52, 56 in one half 42 ofthe housing 41 and the housing is closed so that the needles are heldfirmly in place in the respective bores formed by the recesses. Theneedles are then inserted into the body tissue as shown at FIG. 2a . Theneedles are pushed down to the correct depth for injection of the DNAand this is then carried out. After the injection, the needles are thenpushed slightly further down into the body tissue and the electric powersupply V is activated by a foot pedal (not shown) to apply an electricfield via the needles.

After the electric field has been applied, the needles are removed fromthe body tissue and the housing is opened so that the needles can belifted out of the recesses. The housing is then ready to be reused withnew needles.

A third and most preferred embodiment of the invention will now bedescribed with reference to FIGS. 7 to 13. As shown in FIG. 7, thedevice comprises a base 70 which holds two syringe devices 72, 74 and acover 76. The base 70 is capable of sliding relative to the cover 76.This motion simultaneously inserts both the needles 78, 80 of thesyringe devices and drives a gear mechanism (see FIG. 16) to causeinjection of fluid via the needles. This will be described in greaterdetail below.

The base 70 is shown in FIG. 9. It is formed from plastic (for examplepolyvinyl chloride) although it could also be produced in other suitablematerials such as stainless steel (or mouldable plastics). The base 70has a long solid body which is substantially rectangular in plan view. Abottom surface 82 thereof is substantially flat and is adapted to restslidably on an inner surface of the cover 76. A first end 84 of the base70 is adapted to slide forwardly into engagement with the cover 76 andso comprises a contact surface 86 extending upwardly at an acute angle(45 .degree. in the embodiment shown) from an end of bottom surface 82.A chamfer 88 is provided between the angled contact surface 86 and theupper surface 90 of the base 70.

The upper surface 90 of the base 70 is adapted to receive syringedevices 72, 74. The first part 92 of the upper surface 90 extendsrearwardly from chamfer 88 to form a first planar surface which isparallel to bottom surface 82 and extends a short distance (preferablyabout 16 mm or about 6% of the total length of the base 70) rearwardlyof the chamfer 88 end. Contacts 91 for providing electrical power toeach needle are provided on the base, and power may be supplied to thesevia wires connected to any standard plug and socket arrangement. Thecontacts also form a stability arrangement 91 for holding and supportingthe needles during electroporation.

The combined contact and stability arrangement 91 is provided by twohooked metal plates attached to the angled contact surface 86. Thehooked metal plates are electrically connected to wires (not shown)which may supply electrical power from any suitable power supply via theabove-mentioned plug and socket arrangement (not shown). Furthermore, atchamfer 88, springs 89 are provided, the springs also being electricallyconnected to the above-mentioned wires. The springs 89 serve to pressthe needles 78 and 80 against their respective contacts 91, therebyensuring electrical connection.

Beyond first part 92, a pair of parallel syringe holding grooves 94, 96extending in the direction of the longitudinal extent of base 70 areprovided. The grooves 94, 96 have external side walls which are coplanarwith and form part of the side walls 96, 100 of base 70 and have acentral wall 102 separating the two grooves. The external side walls andcentral wall have straight sides and extend above the level of firstpart 92 of upper surface 90 (preferably by about 9 mm). Further thegrooves 94, 96 are formed with semi-circular bases having a radius ofcurvature of 3.3 mm and the lowest part of the grooves is located abovethe first part 92 of upper surface 90 (preferably by about 2 mm). Thegrooves 94, 96 extend over a distance of about 2 to 3 times the lengthof first part 92 of upper surface 90 (preferably over about 16% of thetotal length of the base or about 41 mm).

Rearwardly of the parallel syringe holding grooves 94, 96, a secondplanar surface 104 extends parallel with the bottom surface 82 and onthe same level as the lowest part of grooves 94, 96. The second planarsurface has a similar length to the parallel syringe holding grooves94,96 (and preferably extends over about 13% of the total length of thebase or about 33 mm).

Rearwardly of the second planar surface 104, a notch 106 is cut out ofthe base 70 extending across the base (i.e. perpendicular to thelongitudinal extent thereof). The notch 106 has straight side edges 108,110 and is cut out to a level below the second planar surface 104(preferably by about 7.5 mm). The notch preferably has a dimension ofabout 3 mm in the longitudinal extent of the base 70).

At the side of notch 106 facing away from the second planar surface 104,a third planar surface 112 extending parallel to the bottom surface 82is provided at a level above the base of notch 106 but below secondplanar surface 104. (The third planar surface 112 is preferably at alevel about 3 mm below second planar surface 104). The third planarsurface 112 preferably extends over about 31% of the total length of thebase or over a distance of about 79 mm.

Immediately rearwardly of the third planar surface 112 a fourth planarsurface 114 extends parallel to the bottom surface 82 and above thethird planar surface (preferably about 14.3 mm above the third planarsurface). A straight edge 116 extending perpendicular to thelongitudinal direction joins the third and fourth planar surfaces toeach other.

The second end 118 of the base 70 comprises a planar surface extendingperpendicular to the longitudinal extent and joining the fourth planarsurface 114 to the bottom surface 82.

A groove 120 with straight edges is cut out from the upper surface 90 ofthe body of the base 70, the groove extending longitudinally along thecentre of the base from the second end 118 thereof to a point within thethird planar surface 112 close to the notch 106 The groove 120 has aflat bottom which is about 4 mm below the level of the third planarsurface 112. The groove is about 4.1 mm wide.

An aperture 122 is cut through one side of the base 70 underneath thefourth planar surface 114 to the groove 120 to form a longitudinallyextending guide in which a pin may slide. The aperture is preferably 4.2mm high and about 29 nun long, is centred about 4.7 mm below the fourthplanar surface 114 and extends from about 8 mm from the second end 118of the base 70.

A circular aperture 124 is cut through the base 70 to the groove 120 andis located on the same side of the base 70 as aperture 122 underneaththe fourth planar surface 114. The aperture 124 is centred on a pointabout 8 mm from the straight edge 116 joining the third 112 and fourth114 planar surfaces arid about 5.3 mm below the fourth planar surface114. The aperture 124 has a diameter of about 3 mm.

A second circular aperture 126 is cut through the base 70 on the otherside from and centred on the same point as the circular aperture 124.The second circular aperture 126 has a diameter of about 10 mm.

A gear wheel 148 on an axle 150 is mounted externally of the base 70 bypassing the axle through the first circular aperture 124 and thenthrough the second circular aperture 126 and securing the axle using anut on the other side of the base 70. In use, the base is movedforwardly relative to the cover and the gear wheel 148 engages on a rack146 provided on toothed member 170, or on a toothed track provided onthe cover to cause the gear wheel 148 to rotate. The gear wheel isadapted to engage with a smaller gear wheel 149 also mounted on the axle150 which drives injection of fluid from the two syringes mounted on thebase by a further gear-rack mechanism 171. As shown in FIG. 18a , aspring 151 is mounted on the axle 150 between the large gear wheel 148and the smaller gear wheel 149. The spring 151 enables a one-way gearmechanism by virtue of which large gear 148 drives small gear wheel 149when rotating in a first direction but does not drive the small gearwheel when rotating in the opposite direction. This will be described infurther detail below.

A lever, 159 is provided on base 70 at the end 118 thereof which can bepulled out from the base to shorten the length by which the needles canproject beyond base 70.

As stated above, the base 70 is adapted to be received within a cover 76as shown in FIG. 7. The cover 76 is shown in greater detail in the crosssectional side view of FIG. 13. The cover is again a solid body whichcould for example be made of polyvinyl chloride.

The cover 76 has a first side wall 128 shaped to cover substantially allof the base 70. The side of the cover opposite the first side wall 128is open to allow access to the base 70 when it is mounted in the cover.A first end 134 of the cover is shaped to cooperate with the first end86 of the base 70, i.e. it extends upwardly at an acute angle(45.degree. in the embodiment shown) away from the bottom of the cover.The opposite end of the cover is open such that the base 70 projectsbeyond the open end when inserted in the cover in use.

On the bottom of the cover 76 extending outwardly from the first sidewall 128 is a planar support surface 130 which extends across the fulllength and width of the cover so as to receive the bottom surface of thebase thereon. An L-shaped guide groove 132 is provided in the supportsurface 130 extending from the open side of the cover across the supportsurface perpendicular to the longitudinal direction approximately to thecentre of the support surface and then extending in the longitudinaldirection towards the first end of the cover. This guide groove 132 isadapted to receive a pin 136 attached to the bottom surface 82 of base70 in use and a user moves the base forwards and backwards relative tothe cover by manually moving this pin 136 in the groove 132. The pin 136and guide groove 132 arrangement has the advantage that the base cannotfall out of the cover in use.

Further supports which hold the base 70 in place within the cover 76 inuse are provided projecting from the first side wall 128 to the otherside of the cover. These supports project both over the first end of thecover and along the top or upper edge thereof (forming parts 134 and 138respectively). These are dimensioned so that gaps are left between theupper support 138 of the cover and various parts of the base 70 in use.A flat portion 140 extends perpendicular to the longitudinal extent ofthe cover between the sloping part of the first end 134 and the upperedge 138 of the cover. This flat portion is provided to be easily placedon the skin of a subject for injection and two apertures 142, 144 areformed through it to allow two needles supported adjacent one anotherabove the base 70 to pass through the cover for insertion.

A toothed track 146 is provided on the upper support 138 to engage withthe gear wheel 148 mounted on base 70 in use.

A stopping member 164 including a projection for engaging with the openend of cover 76 is mounted on base 70 by a screw 166 engaging in thelongitudinal aperture 122. The distance that the base can move withinthe cover (and hence the maximum achievable depth of needle insertion inuse) can be adjusted by moving the stopping member 164 relative to thebase 70 by sliding the screw 166 in the aperture 122. The longitudinalaperture 122 may be provided with a scale to indicate the maximum depthof needle insertion enabled at respective positions of screw 166.Alternatively, the scale could be provided on the base 70 itself to beread off against a point on the stopping member 164.

In use, the base 70 and cover 76 are separated. The gear wheel 148 isthen pushed right back on the toothed track or rack 146 until itdisengages therefrom. This enables the later placement of full syringesinto the base without any fluid being spilled. Either one or bothsyringes are then filled with fluid (this depending on the treatmentdesired). The two syringes 72 and 74 having barrels 152, 154 are themounted in base 70 such that the needle ends 156, 158 extend beyond theend of the base and the ends of their piston rods 160, 162 abut againsta pushing mechanism 171 driven by the small gear wheel 149.

One of the two syringes contains DNA or another substance for injectioninto the person or animal to be treated. The other syringe may be emptyand be used solely to act as an electrode during the subsequentelectroporation process or it may be full of DNA or other fluid forinjection in the same manner as the first syringe. The syringes are heldagainst axial movement relative to the base 70 by annular projections157 provided on the syringes which are received in the notch 106 in base70 in use. The syringes are held against movement in the directionperpendicular to the axial direction by the grooved 96, 98 which extendupwardly on either side of each syringe when fitted in the base.

The base 70 is inserted into cover 76 through the open side thereof, thepin 136 in the bottom of base 70 sliding along the groove 132 in adirection perpendicular to the longitudinal extent of the base until itreaches the bend in groove 132. Four adjustments are then made. Firstly,the lever 159 is adjusted so that the needles only stick out of thecover by a distance corresponding to the fat thickness of the subject tobe treated (i.e. to the depth of initial needle insertion before fluidinjection commences). Next, the base 70 is pushed forward within thecover 76 to reach the maximum desired needle insertion depth and thescrew 166 is locked within aperture 122 at this point. The base is thenpushed back towards the lever 159 and the further gear-rack mechanism171 is pushed forward against the syringe pistons ready for injection.The device is then ready to start the injection process as shown in FIG.14.

Next, the flat portion 140 of the cover 76 is placed on the skin of asubject to be treated and the base 70 is moved towards the first end 134of the cover by pushing the base in that direction using the pin 136. Bymoving the base 70 forward, the needles are moved towards and thenthrough the apertures 142, 144 in the cover 76 so that they penetratethe skin of the subject to be treated. The device at this position isshown in FIGURE and as can be seen, the gear wheel 148 engages toothedtrack 146.

To cause synchronised needle insertion and fluid injection, the pin 136is then manually pushed further forward in the groove 132 thus movingthe cover 76 back towards the stopping member 164 and hence insertingthe needles to a depth determined by the relative position of thestopping member while causing gear wheel 148 to rotate. The rotation ofgear wheel 148 causes the smaller gear wheel 149 to rotate also thuspushing in the piston rods into the syringes such that fluid is injectedgradually through the needles over the depth of insertion of theneedles. FIGS. 16 and 17 respectively show the device halfway throughneedle insertion and when insertion has been completed.

After injection has been completed, an electric field is activatedthrough a current supplied through the needles. The device includes, oris used in conjunction with, a power supply or pulse generator and acontrol box (not shown) through which the level of the voltage suppliedfor electroporation can be varied. Further, the amount of currentdelivered through the needles during electroporation can be measured.Similarly, other characteristics such as electrical resistance can alsobe measured and recorded either before or after the application of thevoltage pulses. The needles are subsequently withdrawn from the subjectbeing treated, by moving pin 136 back in groove 132 to pull the baseback from the cover such that the needles are clear of the cover and thebase is then removed from the cover through the open side thereof. Theneedles can then be lifted away from the base and replaced by newsyringe devices when a new treatment is required. In an alternativewhere the device is set up for multiple injections with a multi-dosesyringe, the needles are retained in the base and further injections canthen be carried out.

In an alternative embodiment of the device, automatic needle insertionand injection can be achieved by respective servo motors. This has theadvantage that the depth of needle insertion can be varied using acontrol for the servo motors.

When treating a human or animal subject, it is important that injectionof fluid is commenced and stopped at suitable needle depths. The depthsat which injection should be started and stopped will vary from subjectto subject depending on the thickness of the superficial fat layer andmuscle of the subject. Thus, the device, power supply or control box mayinclude means for measuring the change in impedance between the needlesof the two syringes during insertion. This change in impedance providesan indication of when the needles have moved into the desired type ofbody tissue for fluid injection to commence as the impedance measuredbetween the needles will be different for different types of bodytissue. In an alternative embodiment of the device, an ultrasoundtransducer can be provided on the tip of a needle to measure the depthof the muscle below the tip of the needle and so determine wheninjection should be commenced.

The device described above could be used with standard syringes as areknown in the art. However, it could alternatively be used with prefilledvials or barrels containing the treatment fluid in single or multipledoses and adapted to be connected to injection needles. This has theadvantage that the user does not need to fill a syringe with theappropriate dose from a bottle of medicament/solution.

A single-dose barrel could be used for treating humans but a multipledose barrel could, for example be used to treat a whole herd of farmanimals with a single needle.

The syringes or barrels for use with the device according to theinvention could be identified by unique bar-codes or other identifiers.The bar-codes could be stored in an electronic controller for the deviceand could be linked to the patient protocol or animal number. Ideally,an iris-scan or ID tag could be used to identify a patient and a DNA IDcode could be provided on the fluid vessel (normally in the form of abar-code). The patient protocol could be automatically retrieved from acomputer when the bar-code on the fluid vessel was read prior to use,leading to great savings in time and effort in clinical situations. Datasuch as the level of current applied during electroporation, and theamount of DNA or fluid injected could also be stored electronically withthe patient protocol. This would enable improved tracking of patientrecords.

A test of the device of the third embodiment has been carried out onsheep. The device of FIG. 7 was used to distribute DNA encoding SEAP orbeta-galactosidase in body tissue. Electroporation was carried outimmediately after insertion of the needles and injection. To administerSEAP for measurement in serum, three sheep were sedated and shaved atone side of the rear. Local anesthetics were applied in a half circlearound the site of treatment. The device was loaded with syringescontaining DNA encoding human serum alkaline phosphatase (SEAP). Onedose consisted of 33 .mu.g DNA in a total of 200 .mu.l. After insertionand injection, current was applied through the needles (400 .mu.secpulses, 1000 Hz, repeated 7-10 times, 35-60 V/cm). Serum samples werecollected 7 days later and measured for SEAP expression by the methoddescribed by Chastain in Journal of Pharmaceutical Science 90 474-484(2001).

To transfect muscle tissue with eDNA encoding beta-galactosidase(.beta.-gal), in order to assess .beta.-gal expression, one sheep wastreated as described above. The device was loaded with syringescontaining DNA encoding beta-galactosidase, and one dose consisted of 40.mu.g DNA in a total of 200 .mu.l. Muscle biopsies were taken 3 dayslater and beta-galactosidase activity was visualised by the method ofSanes et al. Development 113 1181-91 (1991).

The results of the test are shown in FIG. 19 which shows the amount ofSEAP in serum and FIGS. 20a and 20b which show the beta-galactosidase inmuscle. The sheep were given 3 different doses of DNA encoding SEAP asshown in FIG. 19. As shown in FIGS. 20a and 20b , the method gave evendistribution of DNA which in turn gives better and more reproducibleaccessibility to target cells and thereby better transfection.

As a further test of the third embodiment of the invention, experimentswere conducted to measure the resistance between the needles followinginsertion and optionally injection. Sheep were used for the purpose. Thesyringes were filled with saline, mounted on the base unit of the deviceand the cover applied. The needles of the device were inserted into themuscle with or without injection of saline and the resistance measuredby use of a control box.

The resistance in muscle without saline injected was measured at 332ohms, with a total of 100 microliter saline injected the resistance was291 ohms and resistance in muscle with a total of 400 microliter salineinjected was 249 ohms.

In a yet further test, the third embodiment was also tested upon a humanvolunteer in order to assess whether the use of this device would betolerable in humans and whether local anaesthesia would be necessary.

The syringes were filled with saline and mounted in the device. Thedevice was pre-set to allow penetration through the skin (3 mm) and afurther 1 cm of needle insertion with concomitant injection of saline.

The skin of the leg muscle was disinfected and the needles were insertedinto the skin. Then the needles were further inserted, and salineinjected, into the muscle by pushing the knob (136). When the needleswere in place, the electroporation was performed. The pulse given lastedfor 20 ms. The voltage was changed successively from 10 V to 70 V (in 10V steps), with new insertions and injections of saline each time.

At the highest voltage the current delivered was around 240 mA. Theresistance in the muscle tissue was around 300 ohms (within the samerange as seen in sheep).

The description from the volunteer was that the injection and insertionwere without any pain. The electrical stimulation was rated asunpleasant but not painful. Some stiffness in the treated area wasexperienced 1-3 hours after the treatment. The stiffness was lesspronounced than after physical exercise. No anesthesia was used orconsidered necessary in this case although a local anesthesia may bebeneficial if larger areas of the muscle are to be treated.

The embodiments of the electroporation device described above arepreferred embodiments only to which various modifications could be made.For example, the sheaths in the first embodiment could be made of amaterial other than TEFLON® and the apertures in them could be providedin a different pattern. Further, although the device has been describedas including a syringe arrangement to which the needles are connected,it will be appreciated that this need not be an integral part of thedevice. Thus, in an alternative embodiment, the needles in the devicecould be left free to be connectable to a fluid delivery system such asa syringe in use.

Further, although the needles of the device of the second embodimenthave been described as being attached to a syringe arrangement, it willbe appreciated that the needles and syringe part could be providedseparately. Further, although the housing has been described as beingformed in two halves each having two recesses formed therein, it will beappreciated that it could be formed by any number of parts which allowedthe needles to be removed from the housing without pulling out in theaxial direction. Further, it could be adapted to hold any desired numberof needles. Thus, the scope of the invention is not limited by theembodiments of the device as described above but rather is defined bythe scope of the appended claims.

What is claimed:
 1. A method for delivering an active agent into a bodytissue, comprising: inserting at least first and second hollow needlesin the body tissue to a first desired depth, wherein first and secondsyringes are connected respectively to the first and second hollowneedles; advancing the at least the first and second hollow needles fromthe first desired depth to a second desired depth in the body tissue;wherein the advancing step comprises rotating a gear engaged with arack, thereby causing the rack to advance first and second pistons,respectively into the first and second syringes, thereby injecting fluidfrom the first and second syringes and through the first and secondhollow needles and gradually into the body tissue at a rate that isuniform over time while the first and second needles are being insertedinto the body tissue from the first desired depth to the second desireddepth; and electroporating cells of the body tissue during or after thefluid has been injected, thereby delivering the active agent into thebody tissue.
 2. The method of claim 1, wherein the injection commenceswhen the at least the first and second hollow needles reach the firstdesired depth in the body tissue and stops when the at least the firstand second hollow needles reach the second desired depth in the bodytissue.
 3. The method of claim 1, further comprising: coupling the firstand second hollow needles and the first and second syringes to a baseprior to the inserting step; wherein the advancing step comprises movingthe base forward relative to a cover, such that a pin of the basetravels along a guide groove defined in the cover.
 4. The method ofclaim 3, wherein the advancing step comprises driving the pin along theguide groove.
 5. The method of claim 4, wherein driving the pin isperformed manually.
 6. The method of claim 4, wherein the coupling stepcomprising positioning the first and second syringes within respectiveparallel grooves formed in the base.
 7. The method of claim 6, wherein:the cover has a bottom, a flat portion extending from the bottom, and asupport frame, wherein the guide groove is defined by the support frameand extends from the flat portion; and during the advancing step, thebase slideably engages an inner surface of the cover to move laterallybetween the bottom and the support frame.
 8. The method of claim 7,further comprising contacting the flat portion against a tissue surfaceprior to the inserting step.
 9. The method of claim 7, wherein anadditional rack is disposed on the cover, and an additional gear isrotatably connected to the gear, such that rotating the additional gearduring the advancing step while the additional gear is engaged with theadditional rack rotates the gear, thereby causing the rack to advancethe first and second pistons.
 10. The method of claim 9, wherein thegear and the additional gear are rotatably coupled in one-way fashionsuch that the additional gear drives the gear when rotating in a firstdirection and does not drive the gear when rotating in a seconddirection.
 11. The method of claim 7, wherein the at least the first andsecond hollow needles advance through apertures defined in the flatportion during the advancing step.
 12. The method of claim 3, furthercomprising adjusting a position of a stopping member mounted on thebase, wherein a distance between the stopping member and the coverdefines a distance that the base can move laterally within the cover.13. The method of claim 12, wherein the distance between the stoppingmember and the cover corresponds to the second desired depth.
 14. Themethod of claim 12, wherein the adjusting step comprises rotating adrive screw coupled to the base and the stopper.
 15. The method of claim3, further comprising adjusting a position of a lever mounted to thebase to set an initial distance that each of the at least the first andsecond needles protrudes through the apertures.
 16. The method of claim15, wherein the initial distance corresponds to the first desired depth.17. The method of claim 1, further comprising sensing, with a moveablecontact, when the at least the first and second hollow needles have beeninserted to the second desired depth.
 18. The method of claim 17,further comprising moving the contact relative to the at least the firstand second hollow needles and the fluid delivery assembly to adjust thesecond desired depth.
 19. The method of claim 1, further comprisingmeasuring a change in impedance between the first and second hollowneedles during the inserting step, thereby determining when the firstand second hollow needles reach the first desired depth.
 20. The methodof claim 1, wherein the electroporating step comprises deliveringelectric current to the at least the first and second hollow needles.